JP2001514866A - Cryoprotectant removal method and apparatus - Google Patents

Abstract

  (57) [Summary] A method and apparatus for removing cryoprotectants in cryopreserved samples using turbulence and convective dispersion around cells in a cryopreserved primary container is described. Convective dispersion is provided through the combined action of gravity and the pulsatile transmembrane flow of buffer through a hole in a container made of a suitable membrane material. Using mechanical force to assist the cryoprotectant removal process reduces the actual removal time while allowing the benefits of slow cryoprotectant removal to be retained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION

The present invention relates to a mechanically assisted method and an apparatus therefor for removing a cryoprotectant from a suspension of cells in need of cryoprotection.

[0002]

BACKGROUND OF THE INVENTION

Cryopreservation is a procedure for preparing a suspension of cells or a group of cells (eg, embryos) for storage. This procedure usually involves adding a cryoprotectant to the cells to be stored,
This includes cooling the suspended cells, prolonged storage of the cell suspension at temperatures below about -80 ° C, warming the cells to normal cell temperatures, and removing cryoprotectants from the cells. Cryopreservation of sperm or other cells from normal mammals is a seemingly simple method and is successful despite certain serious obstacles. This success relies on the use of one or more cryoprotectants for particular procedural parameters.

[0003] The overall cryoprotection procedure therefore generally involves the following steps: cryoprotectants are added to prepare a cell suspension for cryopreservation, and the individual units are placed in vials or "straws". Cooling at an appropriate rate (sometimes called "freezing"); long-term storage of cell suspensions at temperatures below -80 ° C, often -180 to -196 ° C; Feeding the mediator or user at a low temperature; warming (usually called "thawing") to the normal cell temperature at the appropriate rate; cryoprotectant and Controlled removal of other media or other regulators. The goal is not only to keep the cells alive (keeping them viable), but also for all cellular attributes, such as normal lifespan, oxygen carrying capacity (
Optimizing the maintenance of fertility (especially in the case of red blood cells) and fertility (especially in the case of sperm or oocytes), for which the cells are preserved aside.

[0004] Despite the cell type, species of origin or the various protocols used, prior art cryopreservation protocols traditionally have a mortality (or worse) of about 30% of the cells stored. Results. Many cells traditionally did not survive cooling and rewarming, but surviving cells also suffered further damage during the removal of intracellular cryoprotectants. Causes of damage include inadequate rate of temperature change during cooling and reheating, formation of ice crystals, reduced temperature itself, toxicity due to high concentrations of solutes in and around cells, cryoprotection used. The nature and concentration of the agent (s), the rate at which the cryoprotectant is added to and removed from the cells, and some or all of the other lesser known but empirically obvious factors It is possible.

[0005] Cryopreservatives are molecules that allow a substantial proportion of cells to survive the freeze-thaw cycle and maintain normal cell function. Cryopreservatives that cross the cell plasma membrane and thus act both intracellularly and extracellularly are termed osmotic (penetrating) cryopreservatives. Non-permeable cryoprotectants only work extracellularly. Glycerol is the most effective osmotic cryoprotectant for certain types of cells and sperm from most species. Glycerol is less toxic as compared to other osmotic cryoprotectants, such as ethylene glycol, propylene glycol and dimethyl sulfoxide. All osmotic cryoprotectants pass through cell membranes at a slower rate than water, and each of these rates itself is temperature dependent. Non-permeable cryoprotectants include, for example, proteins (eg, milk or egg proteins used in mammalian sperm); saccharides, such as lactose, fructose, raffinose or trehalose); synthetic polymers, such as methylcellulose; and amide compounds. . Most osmotic cryoprotectants, such as glycerol, act as solutes (and also cause osmotic flow of water) and act as water-miscible solvents (dissolve salts and sugars). All non-penetrable cryoprotectants are solutes or colloids and cannot themselves act as solvents. Both water and glycerol, like other solvents, pass through the cell membrane and eventually equilibrate at the same concentration in all internal structures, resulting in the same intracellular and extracellular concentrations.

[0006] The solute role of osmotic cryoprotectants is believed to cause damage by water-induced osmotic flow. However, the solvent action is beneficial because osmotic cryoprotectants, such as glycerol, have a much lower freezing point than water. In the presence of glycerol, the unfrozen solvent mixture is higher at any given temperature than when water is the only solvent. Therefore, at any given temperature, there is more space for cells and a lower concentration of solute in the channel of unfrozen solvent (the same amount of solute is contained in more liquid). This phenomenon occurs both inside and outside the cell. Furthermore, the presence of glycerol probably reduces the formation of microcracks in ice, which in turn minimizes damage to cells. Impermeable cryoprotectants, such as sugars and lipoproteins, are typically present at relatively high concentrations. They typically act by modifying the plasma membrane to better withstand temperature-induced damage, or simply act as solutes that lower the freezing point of a solute / solvent combination.

[0007] Conventional procedures for preserving many cells have been, not only for a long time, but also recently, despite the warning that osmotic cryoprotectants should be added slowly.
It involves the sudden addition of an osmotic cryoprotectant, such as glycerol, to the cell suspension. Similarly, the benefits of slowly removing an osmotic cryoprotectant from cells are well known. Damage associated with the rapid addition or removal of osmotic cryoprotectants can result in significant changes in cell volume due to the rapid movement of water, and the formation of irreversible "rips" in the plasma membrane. It is a direct result. The slow removal of cryoprotectant from within thawed cells has generally not been used because of lacking or lacking a convenient approach to achieve it.

[0008] Depending on the number of cells required for a functional "unit" after thawing, cells are traditionally packaged as individual units using glass ampules, plastic vials, plastic straws or appropriately sized plastic bags. Is done. All of these packages require removal of the cell suspension from the primary container before slowly removing the cryoprotectant. Alternatively, the technology disclosed by Hammerstedt et al.
U.S. Pat.Nos. 5,026,342 and 5,261,870, both incorporated herein by reference) disclose an open hole in a special membrane formed to provide a primary container that allows liquid exchange across the membrane. While leaving the cells in this primary container,
Allows the cryoprotectant to be removed slowly. Although effective, this method can take up to 2 hours to reduce the intracellular cryoprotectant concentration to the desired level because this approach is diffusion limited.

[0009] Apart from the widely practiced serial dilutions used to address this problem, the slow removal of cryoprotectant from cells in suspension requires conventional dialysis after the suspension is thawed. This can be achieved by placing in the membrane and suspending the dialysis unit in a large volume of salt solution. Alternatively, special pore-closing vessels, such as those disclosed in the U.S. patents incorporated herein by reference above, may open the pores of the membrane vessel after melting to allow the movement of molecules across the membrane. Can be processed.
In both cases, the movement of cryoprotectant and water through the membrane of the primary vessel is through diffusion and is “down” along the concentration gradient (ie, completely away from the locus of highest concentration). ing. Such a diffusion-based method effectively limits the rate at which the composition of the medium directly surrounding the cells is changed, as the water and cryoprotectant diffuse in and out of the primary container membrane, respectively. As a result, this limits the rate of movement of water and cryoprotectant across the cell membrane to a flow rate that does not damage the cells. The reliability of simple diffusion through a membrane vessel is not commercially acceptable1
It takes a long time of ~ 2 hours.

[0010] An alternative auxiliary cryoprotectant removal method employs continuous flow (flow) centrifugation, such as that discussed in US Pat. No. 4,221,322. This requires a transfer from the original cryoprotectant packaging, thus providing a chance for contamination and requiring extra processing.

Thus, one or more cryoprotectants are added to and removed from cells or groups of cells that need to be cryopreserved, avoiding cell damage, possible contamination and / or long processing times in the prior art. There remains a need for a way to do that.

[0012]

Summary of the Invention

To satisfy this need, the present invention relates to gravity and primary containers made from suitable membrane materials, such as U.S. Pat.
No. 6,342 and 5,261,870 disclose turbulence and convective dispersion around cells in a cryopreserved primary vessel, provided through the combined action of the pulsatile transmembrane flow of buffer through the pores. Used to embody methods and devices for removing cryoprotectants in cryopreserved samples. By using mechanical force to assist the cryoprotectant removal method, the benefits of slow cryoprotectant removal are retained while the transfer from the primary container is not performed, while the actual removal The time has been significantly reduced,
And sterility is maintained.

[0013]

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for the pulsation of gravity and buffer through a hole in a primary container made of a suitable membrane material, such as those disclosed in U.S. Pat.Nos. 5,026,342 and 5,261,870, which are incorporated herein by reference. And apparatus for removing cryoprotectants in cryopreserved samples using turbulence and convective dispersion around cells in a cryopreserved primary vessel, provided through the combined action of selective transmembrane flow Embodied. By using mechanical force to assist the cryoprotectant removal process, the benefits of slow cryoprotectant removal are retained while the actual removal time is significantly reduced.

[0014] Due to biohazard and sterility considerations, the means that provide mechanical convenience of cryoprotectant removal are optimally those that do not come into direct contact with the inner surface and contents of the primary container. This is especially true in the case of human cells (eg, sperm, oocytes, embryos, fetal cells, or blood cells), any or all of which are in contact with the technician or other operator handling the container. Should not be allowed. The following description describes the use of a combination of gravity, turbulence and convective dispersion in separate vessels formed according to U.S. Pat.Nos. 5,026,342 and 5,261,870 to rapidly reduce the concentration of cryoprotectant. Provide two forms of the novel device.

For certain types of cells (eg, red blood cells) intended for injection into the vascular system or other body compartment, it is desirable to maintain sterility throughout the freeze-thaw and cryoprotectant removal processes. However, it is difficult at present. The cryoprotectant cannot be removed while the cells are in the original blood cell storage bag. For this reason,
The FDA (Food and Drug Administration) limits the time interval between thawing and deglyceroling human red blood cells and injecting them into humans to 24 hours. The main problem is that the thawed cells need to be transferred from the primary freezing container (plastic bag) to a disposable system for removing cryoprotectants, including interconnected sterile buffer containers, centrifuge bowls and cell receivers. That is. Similar problems prevent cryopreservation of other cell types. Thus, in part, the present invention provides a single container in which the primary container stores the cell suspension and the additional components allow rapid removal of the cryoprotectant by the combined action of gravity, turbulence and convective dispersion. Provide one device. The primary container also allows for sterility retention throughout the freeze-thaw and cryoprotectant removal processes and does not dilute the cell suspension.

The method of the present invention represents an improvement over simple diffusion and is best understood in this context. Traditionally, in conventional dialysis machines containing cells and cryoprotectants, or in primary containers made as in the patents incorporated herein by reference,
The cryoprotectant is diffused into the buffer and removed through small holes or other membrane transport gaps in the vial or straw wall. If the buffer flow is continuously provided outside the cell container, the cryoprotectant concentration in the flowing buffer will be kept low and the cryoprotectant in the cell suspension will reduce the porous wall of the container. It is expected to pass "down" along a concentration gradient that traverses into the external buffer.

In contrast to the above, the method of the present invention does not rely on simple diffusion across the vessel wall to remove the cryoprotectant. Furthermore, simple diffusion of the cryoprotectant across the cell plasma membrane is facilitated by convective dispersion in the primary container. This is achieved by properly mixing the process of turbulence and diffusion through the porous membrane caused by transporting the liquid into and through the primary vessel by the pulsating application of pressure. Inside the primary container, turbulence and pulsatile dispersion of the cryoprotectant-free buffer in the cryoprotectant-rich buffer leads to an overall spread of heterogeneity and a relatively dense cryoprotection. The gravity acting on the agent assists in altering the microenvironment near the cell in a controlled manner. The degree of mixing is controlled in part by: the frequency and volume of the cryoprotectant-free buffer pulse entering the primary container;
Buffer inflow and / or infiltration zones for the upper and lower parts of the contents in the primary container (because the cryoprotectant-rich buffer spontaneously settles to the bottom of the container); (Because gravity can affect the redistribution and dispersion of the cryoprotectant-rich buffer in chamber 1); and any movement of the primary container induced by mechanical means. Pulsating flow can be provided with only inward pressure, which allows a small amount of retrograde flow during the interval between pulses. Alternatively, pulsatile flow can be provided using a combination of inward and withdrawal pressures—positive and negative flow—causing both straight and retrograde flow.

The parameters were developed as follows. 30 for removal of cryoprotectant by diffusion
The minimum flow rate to achieve the same cryoprotectant removal requires 0.2 ml / min, assuming a volume of 6 containers (6 x 1 ml) buffer over a period of minutes, and Assuming that half of the time (straight flow) is the actual flow of buffer, a flow rate of 1-3 ml / min will accomplish that task within 12 minutes. Under these conditions, mass transfer is not limited by the transport rate for diffusion of the cryoprotectant from the primary container and then from the cells. Rather, mass transfer from the primary container and mixing of the cryoprotectant-rich and cryoprotectant-free buffers in the primary container occurs via convective dispersion, and the cryoprotectant Move out of the cell by simple diffusion along a concentration gradient that changes at an appropriate rate due to physical dispersion. The convective dispersion transport of the cryoprotectant is approximately equal to that increasing at lower flow rates at a flow rate of about 1.2 ml / min, assuming a membrane area of about 4 cm ² . Is shown. Oscillating flow (start / stop) is used to minimize cell accumulation on the downstream membrane surface and to facilitate the generation of convective dispersion; The vibration frequency may be in the range of minutes, but a frequency of 2 to 40 pulses / minute is often appropriate. In addition, cryoprotectants are less frequent (e.g., 1-2 pulses / min) and less volume (e.g., 0.05
~ 0.1 ml) and change the pulse frequency, pulse volume or both to "ramp-like
It is often advantageous to progressively increase in a ")" manner. This reduces the cryoprotectant concentration in the cell microenvironment at a rate that does not disrupt the cell but facilitates diffusion of the cryoprotectant from the cell within a commercially viable time frame. . Thus, this method provides a proper mix of turbulence, convection and diffusion, and generally widens the heterogeneity of the liquid environment near the cells in a controlled manner. With this approach, 1
The cryoprotectant can be safely removed from the cells in the ml container in less than 15 minutes.

A preferred embodiment of the present invention is a cryoprotectant remover designed to accommodate a separate primary container containing two major surfaces made from a membrane as disclosed in the patents incorporated by reference above. Device. Upon removal of the cryoprotectant, a turbulent flow of a small volume of buffer is provided through the opening in the primary container, followed by convective dispersion in the primary container to provide buffer free cryoprotectant. Slow mixing of the liquid with the cryoprotectant-rich buffer occurs, after which the diluted cryoprotectant is slowly released from the primary container. To induce proper turbulence and convective dispersion and also to minimize the accumulation of cells on the downstream membrane surface, the cryoprotectant-free buffer oscillation (start / stop) ) Flow is used. Therefore, this device provides a proper mixing of the turbulence, convection and diffusion processes, and generally spreads the heterogeneity of the liquid environment near the cells in a controlled manner. This reduces the concentration of the cryoprotectant in the microenvironment of the cell at a rate that does not impair the cell but facilitates the diffusion of the cryoprotectant from the cell.

The simplest device according to the present invention is shown in FIG. A primary container 1 comprising a wall 2 with holes and containing cryoprotectant-containing cells 3 inside the wall 2 is filled with a buffer solution by an external compression diaphragm 4. The external compression diaphragm 4 is driven by any known means, for example by periodic depressurization, to provide a pulsatile compression of the buffer into and out of the primary container 1. The pulsatile pumping inside the buffer and the convective dispersion occurring inside the primary vessel 1 cause the cryoprotectant concentration in the cells 3 and in the primary vessel 1 to increase more rapidly than possible with simple diffusion, but 3 at a speed that still allows. The movement of water into and out of the cell 3 by cryoprotectant is due to diffusion across the cell plasma membrane, and the concentration gradient is steeper than in conventional methods.

A second embodiment of the cryoprotectant removal device of the present invention is shown in FIGS. 2a and 2b. FIG. 2b shows the same structure as FIG. 2a, but showing the disassembled sequence. The same primary container 1 as shown in FIG. 1 is shown at a predetermined position in the divided cubic apparatus 200. The primary container has a pre-sealed hole that is now U.S. Pat.
A porous container opened by the means disclosed in 5,026,342 and 5,261,870 or other means known in the art. The split cubic device 200 includes two chambers 202 and 204, representing an inflow chamber 206 and an outflow chamber 208, respectively. A pair of gaskets 230 seal these chambers around the primary container 1. Chambers 206 and 208 are coupled to inlet port 232 and outlet port 234 as shown. The inflow chamber 206 is connected via an inlet port 232 to a programmed pulse sequence of the desired buffer (each pulse having a specific volume, duration, duty cycle and pulse interval) by a controller known to those skilled in the art. ) Is coupled to a pump 238 capable of delivering the same. Buffer is drawn from the reservoir 240 by the pump 238 and enters the inflow chamber 206, from there through the primary vessel 1, through the outflow chamber 208,
Exits at outlet port 234 for recirculation.

The structures of FIGS. 2a and 2b are made of plastic or polymer prepared by various methods, such as sheeting, injection molding or any other method. Other materials, such as metal or glass or other composites, can also be used. In operation, and by positioning the split cubic device according to the nature of the primary container and the cells therein, the desired orientation of the porous surface of the primary container 1 and the buffer into and through the primary container 1 An optimal direction of liquid flow will be provided. For some cell types, it may be advantageous to direct the buffer stream into the lower region of the primary container 1 so that the gravity forces the cryoprotectant-rich buffer and cryoprotectant in the primary container 1 Mixing with poor buffers can be assisted. By additional movement of the split cubic device 200 in any known manner, but within the defined parameters, the convective dispersion in the primary container 1 is further improved.

[0023] More complex quick cryoprotectant removal devices are available for special applications, for example, in US Pat.
Designed to remove cryoprotectants from human sperm frozen and thawed in CryoCell ^™ according to 6,026,342 and 5,261,870. Systems for this use include (a) a single-use, sterile, integrated unit that contains the necessary buffers, collects waste, and is easily disposable as a biohazard; Full automation via microprocessor control in placing 1; (c) the closure port is opened and then parallel to the membrane surface of primary vessel 1 during slow controlled turbulent transmembrane flow. Liquid flow; (d) controlled addition of desired additives; and (e) commercial features such as intriguing features and minimal cost, plus optional temperature control during warming. It is aimed at preparing for.

In assisting such a specialized system, a third aspect of the present invention is shown in FIG. The entire housing 30 comprises a microprocessor controller 32, a thermionic cooler 34, a piston driver 3 as a mechanical means for producing a pulsating flow.
6 and a receptacle 42 formed as a recess in the housing 30 to accommodate the piston 38, cam drive 40, and disposable insert 44.
The receptacle 42 is temperature-controlled and has a shape in which the disposable insert 44 is fixedly disposed with respect to the piston 38 and the spring-mounted cam driver 40. The disposable insert 44 includes a buffer reservoir 46 and a removable cap 50,
And mounted as a unit in a processing chamber 48 having a sleeve 52 suitable for receiving the primary container 1 according to FIG. The primary container 1 has a sleeve 52
Inside, the membrane surface of the primary container 1 can be turned in a new direction with respect to the flow direction of the buffer solution by rotating 90 ^° about its vertical axis. The spring-loaded cam drive mates with the primary container 1 to facilitate this rotation, which can alternatively be performed manually if no cam drive 40 is present. The vented waste container 54 that maintains sterility is connected to the processing chamber 4 by an exhaust tube 56.
8 and all can be integrated into the disposable insert 44.

In a third aspect of the invention, the disposable insert 44 and its associated structures can be initially sterile, and both sterility and disposability minimize the potential for sample cross-contamination. Or be excluded. Such individual disposable units also always minimize people's biohazard exposure. Piston drive 36
May be a stepper motor, a solenoid driven micropump, or any other means known in the art that exerts a force. In operation, the pulse rate and buffer volume may be constant, more preferably, starting at a pulse rate of 1 to 3 pulses per minute, over a total interval of 2 to 10 minutes, and exceeding 10 pulses per minute. Speed may be increased. The flow is approximately 0.05 per pulse over the same 8-10 minute interval.
An initial flow rate of 0.10.1 ml can be increased to 0.1-0.2 ml per pulse.

The disposable insert 44 can be shaped to be sealed with a tear-off closure (outer layer) as is known in the art. After removing this seal, the cap 50
Is removed, and the primary container 1 is inserted into the sleeve 52 at a position that provides an initial buffer flow parallel to the membrane surface of the primary container 1. Control of the buffer is provided by a microprocessor 32, a piston driver 36 and a piston 38, the microprocessor comprising:
Perform a preset pulse and volume rate. After a predetermined time, the primary container 1 can be rotated (manually or with the guidance of a microprocessor) in the processing chamber 48 at 90 ^° with respect to its vertical axis, optionally with a spring mounted cam drive. Together with the 40 rotations, a vertical (perpendicular) buffer flow is created instead of an equilibrium flow with respect to the porous surface. After the processing of the buffer is completed, a ready light and a ready alarm can be used to ensure that the primary container 1 is being removed for further proper handling. The whole process
It takes less than 30 minutes. Since it only takes a few minutes to replace the disposable insert 44 in the housing 30, even with as few as two or four units as described in the third embodiment, sufficient equipment is needed even for high volume clinical practice. Will provide.

A fourth aspect of the present invention provides an alternative to the rotation of the spring-loaded cam discussed above for redirecting buffer flow from parallel to vertical (perpendicular to the membrane surface). A fourth embodiment of the device of the present invention is shown in FIGS. 4a, 4b and 4c.

For certain types of cells, such as red blood cells, which are desired to be injected into the vascular system or other body compartment, it is desirable to maintain sterility throughout the freeze-thaw and cryoprotectant removal processes. Damage must be minimized. With conventional procedures, this has been difficult. Because, in order to slowly remove the cryoprotectant from thawed cells and to provide a suitable suspension for injection into the patient,
This is because it was necessary to transfer the thawed cells to a countercurrent centrifugal washing device. The fourth aspect of the invention is suitable for addressing this medical challenge. A single unit combines a primary cell container with a membrane (eg, 0.2 μm pore size) that rejects most microorganisms, and a quick cryoprotectant cell removal device; the entire device is a single-use disposable Designed. Thus, the device enables (1) the preservation of sterility throughout the entire process of freeze-thaw and cryoprotectant removal; (2) the combined action of diffusion and convective dispersion advantageously provides a short But provides for the removal of cryoprotectant at a sufficiently slow rate so as not to damage the red blood cells; (3) the device does not dilute the cell suspension, or the cells are centrifuged after thawing. , Which allows the infusion of the processed red blood cells from the container directly into the patient.

The device of the fourth embodiment comprises an outer housing 400 having a support element 402, two membranes 404 providing a major surface whose properties have been selected for a particular application (US Pat. No. 5,026,
No. 342 and 5,261,870), and a supporting screen of sufficient strength and porosity for the same special applications (eg lysis of red blood cells).
408. One major surface of the outer housing 400 is for distributing buffer entering the inflow chamber 502 of the device and for forming the lower surface of the inflow chamber 502 after the bypass tube 504 is closed with the clamp 506. Baffles of one or more types and sizes to assist in directing liquid through
(Deflecting plate) 500 is incorporated. Outflow chamber 508 may be similarly provided with a baffle (not shown). One end of the housing 400 is
02, 404, and 408, through which one or more tubes 510 communicate with the internal chamber, through which cells of suitable composition known to those of skill in the art and in a buffer containing a cryoprotectant contain. A suspension 512 is charged. From another tube 514,
When desired later, additional buffer is used to remove the cryoprotectant, and the waste tube 516 provides a vent when needed. These tubes are made of a suitable "sterile docking" material known in the art. This unit must be
The tube is sealed (radiation gland sterilization). These components can be made of virtually any material, preferably plastic or polymer. Two membranes 4
04 is made from a porous membrane suitable for the application, i.e., the composition, strength, pore size and percentage of pores have been optimized for the particular application. The pores of the membrane are filled with a suspension of cells (eg, red blood cells) in the internal chamber, the contents are cooled to freezing temperature, and the cells are cooled to zero, as described in US Pat. Nos. 5,026,342 and 5,261,870. The material that keeps the pores closed during the time required to re-warm above 0 ° C. and for the desired time thereafter (eg, 5-10 minutes). Material that dissolves from the pores after 3-10 minutes of exposure to a buffer circulating across it, and is initially plugged.

Variations on the fourth aspect include the possibility of the outer housing itself being a balloon or bladder structure, the use of baffles being optional.

A fifth aspect of the present invention is shown in FIGS. 10a and 10b. The reusable housing 1000 incorporates a microprocessor controller 1002, a thermionic cooler 1003, and insulators (not shown). The linear stepper motor 1004 drives a roller or bar 1055 above the bag 1088 containing the buffer 1010, and the roller or bar 1055 also
It is coupled to port 1024 on half of 013. The processing chamber 1013 is a two-part, low-height rectangular tube, each half representing a cylindrical structure with a cubic opening, where both halves are frozen via a locking mechanism. In addition, this double opening grips the primary container 1 as shown in FIG. A port 1025 in the other half of the processing chamber 1013 contains a waste bag 1 containing waste buffer 1014.
089. The primary container 1 is thin (2.5-4.0 mm) rectangular, square with a specific membrane forming the major surface, typically having an internal volume of 0.5-> 5.0 ml, depending on the cell type and species. Alternatively, it is preferably a tubular structure. In a further variation, the primary container 1 comprises a small port sealed with UV glue after loading the cells in the container and before cooling, and a puncture to remove the cells after removing the cryoprotectant. With thin barriers. Alternatively, the primary container 1 is filled with a thin-walled tube about 1 mm in diameter and 10 or 15 mm in length, sealed with a hot glue or adhesive stopper, and the cryoprotectant is removed and protruded from the processing chamber 1013. After removing the closed seal, the cells can be removed.

To assemble the device according to the fifth aspect of the present invention, both halves of the processing chamber 1013 are locked around the primary container 1 by reverse rotation with a finger and the engagement mechanism 1023 shown in detail. . The sterile buffer is then applied to the surrounding pouch 1010 or 1
From 089, both membrane surfaces of the primary container 1 can be brought into contact. In practice, after a few minutes of exposure to the buffer, a hole is opened and the automatic activity of the stepper motor 1004 causes the pulses and flow to similarly controllably change, as described for other aspects of the invention. To cause a controlled buffer circulation.

[0033] The use of the apparatus disclosed herein can be achieved by controlling the flow of buffer both for removal of plugs and for processing the contents of the vessel, and by turbulent flow in the primary vessel. And providing a pulsatile flow of buffer that induces convective dispersion, thereby improving the utility of the vessel containing the obturator membrane.

[0034]

【Example】

 The present invention is further described by the following examples.

[0035]

Example 1 The device according to FIG. 2 was tested as follows. The inflow chamber 206 was pyramidal in shape, as was the outflow chamber 208. Buffer was moved through this device by a solenoid pump delivering 50 or 100 μm per stroke at a stroke frequency of 3 to 120 strokes per minute (computer controlled). The device is such that the primary surface of the primary container 1--two opposing porous surfaces--is horizontal and the inflow chamber is below or above the primary container 1, or the primary surface is vertical. The operation was carried out with the primary vessel 1 arranged.

In the initial test, the rate of glycerol removal from the primary vessel was measured at various conditions (stroke volume, stroke speed, shape and volume of inflow and outflow chambers, starting glycerol concentration, presence or absence of bull or human sperm). Presence). The discharge rate of glycerol from the primary chamber was controlled.

FIG. 5 shows the rate of removal of glycerol from a 1 ml primary container containing 100 million bull spermatozoa in a buffer containing 0.88 M glycerol and is the average of four experiments.
It was evident that the upward flow rather than the downward flow resulted in a lower removal rate and more initial mixing in the primary vessel. In the final test, (a) conventional 1:10 dilution in buffer followed by centrifugation at 350 × gravity for 15 minutes; deglycerolized human sperm; and (b) 1:10 dilution in buffer An eight-stage process followed by centrifugation at 350x gravity for 15 minutes to deglycerolize human sperm, and (c) quick freezing operated using an inflow chamber below the primary container. The deprotected device (RCRD) was used to compare deglycerol with cryopreserved human sperm. Sperm quality assessment for cells deglycerolated in three steps:% of spontaneous motile sperm based on visual or computer-based analysis (using Hamilton-Thorn Research's IVOS system), bound to egg membrane substrate % Sperm (see US Pat. No. 5,743,206) and the penetration of hamster oocytes without zona using procedures known to those skilled in the art. Based on eight replicate experiments, it took 6.8 minutes to reduce glycerol concentration near sperm to <0.03 M, whereas the prior art procedure took longer than 18 minutes to achieve the same result. It took time. FIGS. 6, 7, 8 and 9 show that the apparatus for removing a cryoprotectant shown in FIG.
It shows that it gives equivalent or better quality sperm than the one- or eight-step dilution method of the prior art.

Although the present invention has been described in detail, the present invention should be limited only by the scope described in the appended claims.

[Brief description of the drawings]

FIG. 1 is a side sectional view of a first embodiment of the present invention.

2a and 2b are side sectional views of the second embodiment of the present invention (the latter is an exploded configuration).

FIG. 3 is a side sectional view of a third embodiment of the present invention.

4a, 4b and 4c are plan views, respectively, of a fourth embodiment of the present invention;
It is an end sectional view and a side sectional view.

FIG. 5 is a graphical representation of test data according to the present invention.

FIG. 6 is a graphical representation of test data according to the present invention.

FIG. 7 is a graphical representation of test data according to the present invention.

FIG. 8 is a graphical representation of test data for the present invention.

FIG. 9 is a graphical representation of test data for the present invention.

10a and 10b are side sectional views (detailed insets) of a fifth embodiment of the present invention.

FIG. 11 is a graphical representation of test data for the present invention.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP , KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZWF terms (reference) 4B029 AA08 AA27 BB11 CC01 GA08 GB10 4H011 CA01 CB07 CB08 CC01 CC03 CD06

Claims (12)

[Claims] 1. contacting one or more cells in a suspension with a solution into which a permeable substance contained in the cells can diffuse; and Exerting a repetitive force that causes a wave motion of the solution, wherein the wave motion of the solution alters the removal of the permeable material from the cells; one or more cells; Of removing osmotic substances from a suspension of osmosis. 2. The cells are in suspension in a primary container and the repetitive force is free of at least 0.1 ml / min of permeable material into and through the primary container. 2. The method according to claim 1, wherein a flow rate of the buffer is generated. 3. The method according to claim 2, wherein the osmotic substance is a cryoprotectant, wherein the exertion step is completed within about 30 minutes, and the wave motion increases in frequency or volume over the completion of the process. The method of claim 1. 4. The method according to claim 1, wherein the solution is a buffer. 5. The method according to claim 1, wherein the cell is a sperm or an oocyte. 6. The method of claim 1, wherein said cells are red blood cells or other somatic cells. 7. The method of claim 2, wherein the primary container is a container having two opposing porous surfaces therein. 8. A primary container having an aperture therein; a processing chamber disposed around the primary container and adapted for flow of the solution therethrough; a repetitive force causing a wave motion of the solution to the solution. Pump means adjacent to the processing chamber, suitable for exerting against it; a source reservoir for osmotic free buffer; and a collection receptacle for liquid exiting the processing chamber. Apparatus for implementing the method of claim 1. 9. The process chamber of claim 8, wherein the processing chamber defines a pyramid-shaped, rectangular or column-shaped solution filling cavity surrounding the primary container.
An apparatus according to claim 1. 10. The apparatus according to claim 8, wherein said pump is a vacuum pump. 11. The device according to claim 8, wherein the pump drives a piston or a roller. 12. The apparatus according to claim 8, wherein said processing chamber comprises means for rotating said primary container.